Anthrax is the infection caused by the bacterium Bacillus anthracis. Following deposition of Bacillus anthracis spores in the body, germination results in expression of toxins, including lethal factor, that are fatal due to destruction of cells of the immune system such as macrophages and dendritic cells. The mechanism of lethal factor-induced cell death is due to proteolytic cleavage of the endogenous signaling molecule mitogen activated protein kinase kinase (MAPKK). Compounds that block lethal factor-induced cleavage of MAPKK will block immune cell-mediated fatality of anthrax. In addition, use of anthrax lethal factor protease inhibitors will also block engineered or modified lethal factor that bioterrorists could easily express in other organisms (e.g. adenovirus vectors) with new lethal potential. As an extension of our Phase I-funded discovery of potent, specific inhibitors of lethal factor activity with efficacy in an animal model of lethal factor lethality, we plan to synthesize analogues of our lead molecules with enhanced potency, greater cell penetration and inhibitory activity on other proteases of counter bioterrorist interest including botulinum neurotoxins and furin, the host cellular protease that activates protective antigen. This effort will involve mechanistic and cellular characterization of inhibitors using x-ray crystallographic analysis, site-directed mutagenesis, kinetic enzymology and cell-based efficacy. New compounds discovered in this program will be evaluated in models of efficacy and pharmacokinetics prior to submission of an Investigational New Drug application with the US Food and Drug Administration. These drugs will help the United States counter known and planned threats to our safety.